Uridine-5′-diphosphoglucose (UDPG) activates the P2Y₁₄ receptor, a neuroimmune system GPCR. P2Y₁₄ receptor tolerates glucose substitution with small alkyl or aryl groups or its truncation to uridine 5′-diphosphate (UDP), a full agonist at the human P2Y₁₄ receptor expressed in HEK-293 cells. 2-Thiouracil derivatives displayed selectivity for activation of the human P2Y₁₄ vs the P2Y₆ receptor, such as 2-thio-UDP 4 (EC₅₀ = 1.92 nM at P2Y₁₄, 224-fold selectivity vs P2Y₆) and its β-propyloxy ester 18. EC₅₀ values of the β-methyl ester of UDP and its 2-thio analogue were 2730 and 56 nM, respectively. β-tert-Butyl ester of 4 was 11-fold more potent than UDPG, but β-aryloxy or larger, branched β-alkyl esters, such as cyclohexyl, were less potent. Ribose replacement of UDP with a rigid North or South methanocarba (bicyclo[3.1.0]hexane) group abolished P2Y₁₄ receptor agonist activity. α,β-Methylene and difluoromethylene groups were well tolerated at the P2Y₁₄ receptor and are expected to provide enhanced stability in biological systems. α,β-Methylene-2-thio-UDP 11 (EC₅₀ = 0.92 nM) was 2160-fold selective versus P2Y₆. Thus, these nucleotides and their congeners may serve as important pharmacological probes for the detection and characterization of the P2Y₁₄ receptor.